Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) in mule deer and elk. TSE infectivity can cross species barriers. The fact that BSE has infected humans in Great Britain and concerns that CWD may act similarly in the US underscores the importance of understanding TSE pathogenesis and developing effective therapeutics. The infectious agent of TSE diseases is called a prion and is largely composed of an abnormally refolded, protease resistant form (PrP-res or PrPSc) of the normal, protease-sensitive prion protein, PrP-sen. PrP-res can be deposited in the brain as either diffuse, amyloid negative deposits or as dense, amyloid positive deposits. Amyloid forms of prion disease appear to be less transmissible than non-amyloid forms, suggesting that there is a fundamental difference in how they trigger disease. Furthermore, it is unclear whether or not prion diseases where PrP-res is deposited primarily as amyloid follow the same pathogenic processes as prion diseases where PrP-res is primarily deposited as non-amyloid. We are interested in understanding the molecular mechanisms underlying PrP amyloid formation and have begun to approach this issue using both in vitro and in vivo model systems. This project focuses on: 1) understanding the pathways of PrP amyloid formation and, 2) studying how mutations in PrP influence PrP-res amyloid formation in familial forms of prion disease. Using LC-MS/MS Nanospray Ion Trap Mass Spectrometry, last year we generated the proteomes of prion infected mouse brain tissue which had accumulated PrP-res in either amyloid or non-amyloid forms. In 2012, we have continued processing the high volume of data generated by these experiments. Current analysis of data from this study indicated that a new set of negative controls was necessary and these new controls are currently being processed and analyzed. These studies will provide insight into whether or not amyloid and non-amyloid forms of prion disease differ mechanistically and are expected to be submitted for publication in 2012. Hereditary forms of prion disease are associated with mutations within the PrP gene. One of these mutations is the insertion of extra copies of an eight amino acid motif (octapeptide repeat) into PrP. We have used an in vitro fibrillization model of this hereditary mutation to study how the repeat region influences the formation of both PrP-res and PrP amyloid. Last year, we used standard biochemical techniques and PrP peptides associated with human prion disease to show that the C-terminus affects the structure of the octapeptide repeat region during PrP amyloid formation by influencing the initial folding of PrP-sen, not the actual fibrillization of PrP into amyloid. In 2012, we initiated additional studies to confirm that the octapeptide repeat region acquires structure during fibrillization. Our results have been submitted for publication and are currently under review. These studies may help to determine why the structure of the octapeptide repeat region appears to be strain specific and thus may help to explain the different pathologies associated with different types of prion infectivity. Finally, in 2012 we continued studies using an in vitro system that has been shown to generate infectious prions from recombinant PrP-sen (Science 327: 1132 (2010)). Our current results suggest that this in vitro system favors the formation of abnormal, protease-resistant forms of recombinant PrP that are non-infectious. We expect to submit the results of this work for publication sometime in 2012. Based on our results, we are currently analyzing how abnormal, protease-resistant PrP molecules made in vitro differ biochemically and structurally from those made in vivo.